Tissue culture is a way of getting more cells from the tissue by growing them off of the organism. To do this it is necessary to set up an artificial environment in which the cells will grow. Once the cells have been cultured, they can be used in many different types of studies, including studies on the biochemistry of the tissues and the development of medications.[1]

Steps

Part 1

Preparing to Culture

1

Determine what you will culture. It is possible to culture both plant and animal cells. Sometimes people want to culture tissue that has been obtained directly from a plant or animal. Other times the cells are cultured from a cell line that was previously cultured and stored.[2]

New cultures may be created from taking tissue from an organism. These cells are called a primary culture. They will only divide for so long before they go through senescence.

A continuous cell line has been altered using chemicals or viruses so that the cells will continue to divide indefinitely.

2

Prepare the culture environment. The conditions will be different for different types of cells, but in general, the culture will require:[3]

A controlled temperature. The best temperature will vary on the type of cells you are growing, but it will probably be between 20-45 C.

A controlled pH. The best pH will likely be somewhere between 6.0 and 7.5.

A controlled osmotic pressure

Adequate exchange of oxygen and carbon dioxide

Hormones

Growth factors

A substrate that will provide the cells with the nutrients they need. This includes amino acids, carbohydrates, vitamins, and minerals.

Light. Light is crucial if you are growing plant cells which must photosynthesize.[4]

3

Decide what type of substrate you will provide. Many cell types grow best when they are attached to a substrate. Possible types of substrates include:[5][6]

A solid substrate, also called adherent. Tissues obtained from organs like kidneys are usually adherent because they are fixed in connective tissue.

A semi-solid or monolayer substrate. These types of cultures generally form a layer one cell deep on the bottom.

A suspension culture. Cells of this type do not need to be anchored to the substrate, and will grow while floating instead. Cells from blood are often cultured in suspension.

Part 2

Performing the Culture

1

Follow the manufacturer’s protocols for starting the culture. There are several companies that supply laboratories with materials and equipment for conducting cultures. When you purchase the materials, they will also come with protocols for starting the culture. These protocols will describe exactly how you should transfer the sample that you want to culture into the culture environment. Several well-known companies include:

Qiagen

Fisher Scientific

Thermo Scientific

2

Feed the cells if you are working with mammalian cells. It is generally necessary to replenish the culture media every 2 – 3 days. Follow the manufacturer’s protocols for feeding the cells.[7]

This process should take 20 minutes or less.

If you do not provide sufficient nutrients, the cells that you do have will die.

3

Split cultures that are starting to get crowded. In order to keep the culture growing, you may need to move some of the cells into a new dish with a lower density. This will enable both the original culture and the new one to continue growing.[8]

You can also freeze some of the cultures for future work or to repeat experiments if you have problems with contamination in the future. Follow the provided protocols to determine the temperature for cryopreservation or freezing the cultures.